Notch receptors are widely expressed by hematopoietic precursor cells and the differentiation of hematopoietic precursors is inhibited when these cells are induced to overexpress the constitutively active cytoplasmic domain of Notch1 or when they are exposed to exogenously presented Notch ligand forms. Our studies show that culture of human cord blood cells in the presence of engineered Notch ligand leads to increased numbers of CD34+ precursors, including those able to repopulate immunodeficient mice, and our preliminary data indicate that the dose of Notch ligand plays a critical role in determining hematopoietic stem cell fate. Based on these observations, we hypothesize that the use of different doses and types of Notch ligands can serve to enhance the self-renewal of hematopoietic repopulating cells. To further investigate the role of Notch signaling in hematopoiesis and its effect on stem cell proliferation and differentiation, we will determine: dose-dependent effects of the Notch ligand Delta1 on the growth and differentiation of CD34+CD38- cord blood precursors, in particular on the generation of short- and long-term NOD/SCID repopulating cells (Aim 1);whether different Notch ligand types induce Notch signaling that differentially affects the proliferation and differentiation of CD34+CD38- cord blood cells (Aim 2);and whether induction of different cell fates by different doses or types of Notch ligands results from differences in the constellation of induced Notch target genes (Aim 3). Our overall goal is to define the effects of ligand-induced Notch signaling on hematopoietic stem cell proliferation and differentiation as the basis for methods to manipulate this activity for therapeutic application, in particular, for improving umbilical cord blood transplantation.